The Picornaviridae are a large and diverse family of positive-strand RNA viruses that include a number of important human pathogens, among them hepatitis A virus (HAV), an hepatotropic virus that is the causative agent of acute hepatitis A and the only species in the genus Hepatovirus. As a family, picornaviruses are non-enveloped. Their genomes are encapsidated within icosahedral capsids comprised of 60 copies of each of 3-4 polypeptides. High-resolution structural models have been developed for several pathogenic picornaviruses but not HAV, and its structure remains poorly defined. We have discovered that most HAV particles released by cultured human hepatoma (Huh-7.5) cells are fully enveloped in host-derived membranes. Remarkably, biophysical studies indicate that these enveloped hepatoviruses (eHAV) are also the dominant form of virus circulating in the blood during acute hepatitis A. eHAV particles are fully infectious, yet highly resistant to neutralizing anti-HAV antibodies. These novel observations provide a new view of hepatitis A pathogenesis, and raise important questions about the mechanism by which vaccines provide protection against hepatitis A even when administered 2 weeks after infection. Specific Aim 1 will use quantitative proteomics and immunoelectron microscopy to characterize viral and host-derived proteins present in eHAV particles, as these may provide clues to the origin of eHAV. Specific Aim 2 focuses directly on the biogenesis of eHAV, and will use reverse molecular genetics and RNAi knockdown of multiple host proteins to probe interactions of HAV with ESCRT complexes involved in endosomal sorting and proteins involved in autophagy. Both cellular processes are potentially relevant to eHAV release from cells. Specific Aim 3 addresses the fate of eHAV, focusing particularly on how anti-HAV antibodies neutralize eHAV after its entry into permissive cells, a process that is likely to account for the protection vaccines afford against disease when administered after infection with the virus. A subsidiary aim is to develop a murine model of hepatitis A in which these events can be studied and which can replace existing nonhuman primate models of HAV infection. Although a completely novel finding, membrane hijacking may not be unique to HAV. These studies are thus broadly relevant to other non-enveloped viruses and how vaccine-induced antibodies provide protection against disease. PUBLIC HEALTH RELEVANCE: Hepatitis A virus (HAV) is a common cause of acute viral hepatitis. It is a member of the Picornaviridae family, a diverse group of non-enveloped RNA viruses that includes many human pathogens. We have made the surprising discovery that HAV particles released by infected liver-derived cells in vitro, or circulating in the blood during acue hepatitis A, are packaged within a single-membrane envelope and, although fully infectious, are resistant to antibody-mediated neutralization. This membrane hijacking by HAV has broad implications for other non-enveloped viruses, and for the mechanism by which hepatitis A vaccines protect against disease even when administered 2 weeks after infection.